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Peptidylarginine deiminase 2 citrullinates MZB1 and promotes the secretion of IgM and IgA

ABSTRACT: MZB1 is an endoplasmic reticulum residential protein preferentially expressed in plasma cells, marginal zone and B1 B cells. Recent studies on murine B cells shows that it interacts with the tail piece of IgM and IgA heavy chain and promotes the secretion of these two classes of immunoglobulin. However, its role in primary human B cells has yet to be determined and how its function is regulated is still unknown. The conversion of peptidylarginine to peptidycitrulline, also known as citrullination, by peptidylarginine deiminases (PADs) can critically influence the function of proteins in immune cells, such as neutrophils and T cells; however, the role of PADs in B cells remains to be elucidated. In an unbiased analysis of the human citrullinomeic analysis, we found that MZB1 was preferentially enriched in the pool of citrullinated lung proteins obtained from patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD) compared to those from idiopathic pulmonary fibrosis or chronic obstructive pulmonary diseases. The citrullination of MZB1 was confirmed with mass spectrometry, in vitro citrullination, and binding by phenylglyoxal in the absence or presence of a PAD2-specific inhibitor AFM-30a. Ablation or pharmacological inhibition of PAD2 in primary human B cells attenuated the secretion of IgM and IgA but not IgG or the differentiation of IgM or IgA-expressing plasmablasts, recapitulating the effect of ablating MZB1. In addition, the physical interaction between endogenous MZB1 and IgM/IgA was attenuated by AFM-30a. Taken together, our data confirm the function of MZB1 in primary human plasmablasts and suggest that PAD2 promotes IgM/IgA secretion by citrullinating MZB1, thereby contributing to the pathogenesis of rheumatoid arthritis and RA-ILD.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung

SUBMITTER: Paul Thompson

LAB HEAD: Paul R Thompson

PROVIDER: PXD047044 | Pride | 2023-11-23

REPOSITORIES: Pride

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Project description:Abstract: Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. It is well established that a gonadotropin releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope derived LBT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterize the temporal dynamics of this modification. Our results show that actin and tubulin are rapidly citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. Data shows that 10 nM GnRHa induces citrullination of beta-actin with maximal levels occurring at 10 minutes. The level of beta-actin citrullination is reduced in the presence of the pan-PAD inhibitor bi-phenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa induced actin reorganization in dispersed mouse gonadotrope cells. GnRHa induces citrullination of beta-tubulin with elevated levels occurring at 30 minutes; similarly to beta-actin, this response is attenuated in the presence of BB-ClA. To examine the functional consequence of beta-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LBT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that MT average lifetime increases following 30 minutes of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells. Keywords: gonadotrope, peptidylarginine deiminase, citrullination, cytoskeleton, beta tubulin, beta actin, microtubules

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Peptidylarginine deiminase 2 citrullinates MZB1 and promotes the secretion of IgM and IgA

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